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The α-amylase paralogue Amyrel present in true flies (Diptera Muscomorpha) has been classified as a glycoside hydrolase in CAZy family GH13 on the basis of its primary structure. Here, we report that, in fact, Amyrel is currently unique among animals as it possesses both the hydrolytic α-amylase activity (EC 3.2.1.1) and a 4-α-glucanotransferase (EC 2.4.1.25) transglycosylation activity. Amyrel reacts specifically on α-(1–4) glycosidic bonds of starch and related polymers but produces a complex mixture of maltooligosaccharides, which is in sharp contrast with canonical animal α-amylases. With model maltooligosaccharides G2 (maltose) to G7, the Amyrel reaction starts by a disproportionation leading to Gn − 1 and Gn + 1 products, which by themselves become substrates for new disproportionation cycles. As a result, all detectable odd- and even-numbered maltooligosaccharides, at least up to G12, were observed. However, hydrolysis of these products proceeds simultaneously, as shown by p-nitrophenyl-tagged oligosaccharides and microcalorimetry, and upon prolonged reaction, glucose is the major end-product followed by maltose. The main structural determinant of these atypical activities was found to be a Gly-His-Gly-Ala deletion in the so-called flexible loop bordering the active site. Indeed, engineering this deletion in porcine pancreatic and Drosophila melanogaster α-amylases results in reaction patterns similar to those of Amyrel. It is proposed that this deletion provides more freedom to the substrate for subsites occupancy and allows a less-constrained action pattern resulting in versatile activities at the active site.

In Gram-negative bacteria, the major mechanism of resistance to β-lactam antibiotics is the production of one or several β-lactamases (BLs), including the highly worrying carbapenemases. Whereas inhibitors of these enzymes were recently marketed, they only target serine-carbapenemases (e.g. KPC-type), and no clinically useful inhibitor is available yet to neutralize the class of metallo-β-lactamases (MBLs). We are developing compounds based on the 1,2,4-triazole-3-thione scaffold, which binds to the di-zinc catalytic site of MBLs in an original fashion, and we previously reported its promising potential to yield broad-spectrum inhibitors. However, up to now only moderate antibiotic potentiation could be observed in microbiological assays and further exploration was needed to improve outer membrane penetration. Here, we synthesized and characterized a series of compounds possessing a diversely functionalized alkyl chain at the 4-position of the heterocycle. We found that the presence of a carboxylic group at the extremity of an alkyl chain yielded potent inhibitors of VIM-type enzymes with Ki values in the μM to sub-μM range, and that this alkyl chain had to be longer or equal to a propyl chain. This result confirmed the importance of a carboxylic function on the 4-substituent of 1,2,4-triazole-3-thione heterocycle. As observed in previous series, active compounds also preferentially contained phenyl, 2-hydroxy-5-methoxyphenyl, naphth-2-yl or m-biphenyl at position 5. However, none efficiently inhibited NDM-1 or IMP-1. Microbiological study on VIM-2-producing E. coli strains and on VIM-1/VIM-4-producing multidrug-resistant K. pneumoniae clinical isolates gave promising results, suggesting that the 1,2,4-triazole-3-thione scaffold worth continuing exploration to further improve penetration. Finally, docking experiments were performed to study the binding mode of alkanoic analogues in the active site of VIM-2.

To fight the increasingly worrying bacterial resistance to antibiotics, the discovery and development of new therapeutics is urgently needed. Here, we report on a new series of 1,2,4-triazole-3-thione compounds as inhibitors of metallo-&beta, lactamases (MBLs), which represent major resistance determinants to &beta, lactams, and especially carbapenems, in Gram-negative bacteria. These molecules are stable analogs of 4-amino-1,2,4-triazole-derived Schiff bases, where the hydrazone-like bond has been reduced (hydrazine series) or the 4-amino group has been acylated (hydrazide series), the synthesis and physicochemical properties thereof are described. The inhibitory potency was determined on the most clinically relevant acquired MBLs (IMP-, VIM-, and NDM-types subclass B1 MBLs). When compared with the previously reported hydrazone series, hydrazine but not hydrazide analogs showed similarly potent inhibitory activity on VIM-type enzymes, especially VIM-2 and VIM-4, with Ki values in the micromolar to submicromolar range. One of these showed broad-spectrum inhibition as it also significantly inhibited VIM-1 and NDM-1. Restoration of &beta, lactam activity in microbiological assays was observed for one selected compound. Finally, the binding to the VIM-2 active site was evaluated by isothermal titration calorimetry and a modeling study explored the effect of the linker structure on the mode of binding with this MBL.

Crystal structures of phosphoglycerate kinase (PGK) from the psychrophile Pseudomonas sp. TACII 18 have been determined at high resolution by X-ray crystallography methods and compared with mesophilic, thermophilic and hyperthermophilic counterparts. PGK is a two-domain enzyme undergoing large domain movements to catalyze the production of ATP from 1,3-biphosphoglycerate and ADP. Whereas the conformational dynamics sustaining the catalytic mechanism of this hinge-bending enzyme now seems rather clear, the determinants which underlie high catalytic efficiency at low temperatures of this psychrophilic PGK were unknown. The comparison of the three-dimensional structures shows that multiple (global and local) specific adaptations have been brought about by this enzyme. Together, these reside in an overall increased flexibility of the cold-adapted PGK thereby allowing a better accessibility to the active site, but also a potentially more disordered transition state of the psychrophilic enzyme, due to the destabilization of some catalytic residues.

Summary Trigger factor (TF) is the first molecular chaperone interacting cotranslationally with virtually all nascent polypeptides synthesized by the ribosome in bacteria. Thermal adaptation of chaperone function was investigated in TFs from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli and the hyperthermophile Thermotoga maritima. This series covers nearly all temperatures encountered by bacteria. Although structurally homologous, these TFs display strikingly distinct properties that are related to the bacterial environmental temperature. The hyperthermophilic TF strongly binds model proteins during their folding and protects them from heat-induced misfolding and aggregation. It decreases the folding rate and counteracts the fast folding rate imposed by high temperature. It also functions as a carrier of partially folded proteins for delivery to downstream chaperones ensuring final maturation. By contrast, the psychrophilic TF displays weak chaperone activities, showing that these functions are less important in cold conditions because protein folding, misfolding and aggregation are slowed down at low temperature. It efficiently catalyses prolyl isomerization at low temperature as a result of its increased cellular concentration rather than from an improved activity. Some chaperone properties of the mesophilic TF possibly reflect its function as a cold shock protein in E. coli.

Psychrophiles thriving permanently at near-zero temperatures synthesize cold-active enzymes to sustain their cell cycle. Genome sequences, proteomic, and transcriptomic studies suggest various adaptive features to maintain adequate translation and proper protein folding under cold conditions. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Several open questions in the field are also highlighted.

The online version of this article (doi:10.1007/s00792-016-0851-9) contains supplementary material, which is available to authorized users., Cuproxidases are a subset of the blue multicopper oxidases that catalyze the oxidation of toxic Cu(I) ions into less harmful Cu(II) in the bacterial periplasm. Cuproxidases from psychrophilic, mesophilic, and thermophilic bacteria display the canonical features of temperature adaptation, such as increases in structural stability and apparent optimal temperature for activity with environmental temperature as well as increases in the binding affinity for catalytic and substrate copper ions. In contrast, the oxidative activities at 25 °C for both the psychrophilic and thermophilic enzymes are similar, suggesting that the nearly temperature-independent electron transfer rate does not require peculiar adjustments. Furthermore, the structural flexibilities of both the psychrophilic and thermophilic enzymes are also similar, indicating that the firm and precise bindings of the four catalytic copper ions are essential for the oxidase function. These results show that the requirements for enzymatic electron transfer, in the absence of the selective pressure of temperature on electron transfer rates, produce a specific adaptive pattern, which is distinct from that observed in enzymes possessing a well-defined active site and relying on conformational changes such as for the induced fit mechanism., This work was supported by the F.R.S-FNRS, Belgium (Fonds de la Recherche Fondamentale et Collective, contract numbers 2.4535.08, 2.4523.11 and U.N009.13 to G.F.) and by the Belgian program of Interuniversity Attraction Poles (iPros P7/44) initiated by the Federal Office for Scientific, Technical and Cultural Affairs. T.C. thanks the FCT and FEDER (POFC-COMPETE) for funding through the Investigador FCT Programme (IF/01635/2014) and project EngXyl (EXPL/BBB-BIO/1772/2013- FCOMP01-0124-FEDER-041595) as well as strategic funding via UID/ BIA/04050/2013. F.R., A.G. and A.C. were FRIA research fellows., info:eu-repo/semantics/publishedVersion

Psychrophiles, i.e., organisms thriving permanently at near-zero temperatures, synthesize cold-active enzymes to sustain their cell cycle. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. Considering the subtle structural adjustments required for low temperature activity, directed evolution appears to be the most suitable methodology to engineer cold activity in biological catalysts.

Summary The protein folding process in psychrophiles is impaired by low temperature, which exerts several physicochemical constraints, such as a decrease in the folding rate, reduced molecular diffusion rates and increased solvent viscosity, which interfere with conformational sampling. Furthermore, folding assistance is required at various folding steps according to the protein size. Recent studies in the field have provided contrasting and sometimes contradictory results, although protein folding generally appears as a rate-limiting step for the growth of psychrophiles. It is proposed here that these discrepancies reflect the diverse adaptive strategies adopted by psychrophiles in order to allow efficient protein folding at low temperature. Cold adaptations apparently superimpose on pre-existing cellular organization, resulting in different adaptive strategies. In addition, microbial lifestyle further modulates the properties of the chaperone machinery, which possibly explains the occurrence of cold-adapted and non-cold-adapted protein chaperones in psychrophiles.

Alkaline phosphatases (APs) are commercially applied enzymes that catalyze the hydrolysis of phosphate monoesters by a reaction involving three active site metal ions. We have previously identified H135 as the key residue for controlling activity of the psychrophilic TAB5 AP (TAP). In this article, we describe three X-ray crystallographic structures on TAP variants H135E and H135D in complex with a variety of metal ions. The structural analysis is supported by thermodynamic and kinetic data. The AP catalysis essentially requires octahedral coordination in the M3 site, but stability is adjusted with the conformational freedom of the metal ion. Comparison with the mesophilic Escherichia coli, AP shows differences in the charge transfer network in providing the chemically optimal metal combination for catalysis. Our results provide explanation why the TAB5 and E. coli APs respond in an opposite way to mutagenesis in their active sites. They provide a lesson on chemical fine tuning and the importance of the second coordination sphere in defining metal specificity in enzymes. Understanding the framework of AP catalysis is essential in the efforts to design even more powerful tools for modern biotechnology.

Psychrophilic alkaline phosphatase (AP) from the Antarctic strain TAB5 was subjected to directed evolution in order to identify the key residues steering the enzyme's cold-adapted activity and stability. A round of random mutagenesis and further recombination yielded three thermostable and six thermolabile variants of the TAB5 AP. All of the isolated variants were characterised by their residual activity after heat treatment, Michaelis-Menten kinetics, activation energy and microcalorimetric parameters of unfolding. In addition, they were modelled into the structure of the TAB5 AP. Mutations which affected the cold-adapted properties of the enzyme were all located close to the active site. The destabilised variants H135E and H135E/G149D had 2- and 3-fold higher kcat, respectively, than the wild-type enzyme. Wild-type AP has a complex heat-induced unfolding pattern while the mutated enzymes loose local unfolding transitions and have large shifts of the Tm values. Comparison of the wild-type and mutated TAB5 APs demonstrates that there is a delicate balance between the enzyme activity and stability and that it is possible to improve the activity and thermostability simultaneously as demonstrated in the case of the H135E/G149D variant compared to H135E.

The Antarctic fungal strain Aspergillus glaucus 363 produces cold-active (CA) Cu/Zn-superoxide dismutase (SOD). The strain contains at least one gene encoding Cu/Zn-SOD that exhibited high homology with the corresponding gene of other Aspergillus species. To our knowledge, this is the first nucleotide sequence of a CA Cu/Zn-SOD gene in fungi. An effective laboratory technology for A. glaucus SOD production in 3 L bioreactors was developed on the basis of transient cold-shock treatment. The temperature downshift to 10 °C caused 1.4-fold increase of specific SOD activity compared to unstressed culture. Maximum enzyme productivity was 64 × 103 U kg−1 h−1. Two SOD isoenzymes (Cu/Zn-SODI and Cu/Zn-SODII) were purified to electrophoretic homogeneity. The specific activity of the major isoenzyme, Cu/Zn-SODII, after Q-Sepharose chromatography was 4000 U mg−1. The molecular mass of SODI (38 159 Da) and of SODII (15 835 Da) was determined by electrospray quadropole time-of-flight (ESI-Q-TOF) mass spectrometry and dynamic light scattering (DLS). The presence of Cu and Zn were confirmed by inductively coupled plasma mass spectrometry (ICP-MS). The N-terminal amino acid sequence of Cu/Zn-SODII revealed a high degree of structural homology with Cu/Zn-SOD from other fungi, including Aspergillus species.

Chloride-activated α-amylases contain a noncatalytic triad, independent of the glycosidic active site, perfectly mimicking the catalytic triad of serine-proteases and of other active serine hydrolytic enzymes. Mutagenesis of Glu, His, and Ser residues in various α-amylases shows that this pattern is a structural determinant of the enzyme conformation that cannot be altered without losing the intrinsic stability of the protein. 1H-15N NMR spectra of a bacterial α-amylase reveal proton signals that are identical with the NMR signature of catalytic triads and especially a deshielded proton involving a protonated histidine and displaying properties similar to that of a low barrier hydrogen bond. It is proposed that the H-bond between His and Glu of the noncatalytic triad is an unusually strong interaction, responsible for the observed NMR signal and for the weak stability of the triad mutants. Furthermore, a stringent template-based search of the Protein Data Bank demonstrated that this motif is not restricted to α-amylases, but is also found in 80 structures from 33 different proteins, amongst which SH2 domain-containing proteins are the best representatives. Proteins 2008. © 2007 Wiley-Liss, Inc.

The halotolerance of a cold adapted α-amylase from the psychrophilic bacterium Pseudoalteromonas haloplanktis (AHA) was investigated. AHA exhibited hydrolytic activity over a broad range of NaCl concentrations (0.01–4.5 M). AHA showed 28% increased activity in 0.5–2.0 M NaCl compared to that in 0.01 M NaCl. In contrast, the corresponding mesophilic (Bacillus amyloliquefaciens) and thermostable (B. licheniformis) α-amylases showed a 39 and 46% decrease in activity respectively. Even at 4.5 M NaCl, 80% of the initial activity was detected for AHA, whereas the mesophilic and thermostable enzymes were inactive. Besides an unaltered fluorescence emission and secondary structure, a 10°C positive shift in the temperature optimum, a stabilization factor of >5 for thermal inactivation and a ΔT m of 8.3°C for the secondary structure melting were estimated in 2.7 M NaCl. The higher activation energy, half-life time and T m indicated reduced conformational dynamics and increased rigidity in the presence of higher NaCl concentrations. A comparison with the sequences of other halophilic α-amylases revealed that AHA also contains higher proportion of small hydrophobic residues and acidic residues resulting in a higher negative surface potential. Thus, with some compromise in cold activity, psychrophilic adaptation has also manifested halotolerance to AHA that is comparable to the halophilic enzymes.

The cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis (AHA) is a multidomain enzyme capable of reversible unfolding. Cold-adapted proteins, including AHA, have been predicted to be structurally flexible and conformationally unstable as a consequence of a high lysine-to-arginine ratio. In order to examine the role of low arginine content in structural flexibility of AHA, the amino groups of lysine were guanidinated to form homo-arginine (hR), and the structure-function-stability properties of the modified enzyme were analyzed by transverse urea gradient-gel electrophoresis. The extent of modification was monitored by MALDI-TOF-MS, and correlated to changes in activity and stability. Modifying lysine to hR produced a conformationally more stable and less active alpha-amylase. The k(cat) of the modified enzyme decreased with a concomitant increase in deltaH# and decrease in K(m). To interpret the structural basis of the kinetic and thermodynamic properties, the hR residues were modeled in the AHA X-ray structure and compared to the X-ray structure of a thermostable homolog. The experimental properties of the modified AHA were consistent with K106hR forming an intra-Domain B salt bridge to stabilize the active site and decrease the cooperativity of unfolding. Homo-Arg modification also appeared to alter Ca2+ and Cl- binding in the active site. Our results indicate that replacing lysine with hR generates mesophilic-like characteristics in AHA, and provides support for the importance of lysine residues in promoting enzyme cold adaptation. These data were consistent with computational analyses that show that AHA possesses a compositional bias that favors decreased conformational stability and increased flexibility.

The cold-active α-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (AHA) is the largest known multidomain enzyme that displays reversible thermal unfolding (around 30°C) according to a two-state mechanism. Transverse urea gradient gel electrophoresis (TUG-GE) from 0 to 6.64 M was performed under various conditions of temperature (3°C to 70°C) and pH (7.5 to 10.4) in the absence or presence of Ca 2+ and/or Tris (competitive inhibitor) to identify possible low-stability domains. Contrary to previous observations by strict thermal unfolding, two transitions were found at low temperature (12°C). Within the duration of the TUG-GE, the structures undergoing the first transition showed slow interconversions between different conformations. By comparing the properties of the native enzyme and the N12R mutant, the active site was shown to be part of the least stable structure in the enzyme. The stability data supported a model of cooperative unfolding of structures forming the active site and independent unfolding of the other more stable protein domains. In light of these findings for AHA, it will be valuable to determine if active-site instability is a general feature of heat-labile enzymes from psychrophiles. Interestingly, the enzyme was also found to refold and rapidly regain activity after being heated at 70°C for 1 h in 6.5 M urea. The study has identified fundamental new properties of AHA and extended our understanding of structure/stability relationships of cold-adapted enzymes.

The cold-adapted α-amylase from Pseudoalteromonas haloplanktis unfolds reversibly and cooperatively according to a two-state mechanism at 30°C and unfolds reversibly and sequentially with two transitions at temperatures below 12°C. To examine the role of the four disulfide bridges in activity and conformational stability of the enzyme, the eight cysteine residues were reduced with β-mercaptoethanol or chemically modified using iodoacetamide or iodoacetic acid. Matrix-assisted laser desorption-time of flight mass spectrometry analysis confirmed that all of the cysteines were modified. The iodoacetamide-modified enzyme reversibly folded/unfolded and retained approximately one-third of its activity. Removal of all disulfide bonds resulted in stabilization of the least stable region of the enzyme (including the active site), with a concomitant decrease in activity (increase in activation enthalpy). Disulfide bond removal had a greater impact on enzyme activity than on stability (particularly the active-site region). The functional role of the disulfide bridges appears to be to prevent the active site from developing ionic interactions. Overall, the study demonstrated that none of the four disulfide bonds are important in stabilizing the native structure of enzyme, and instead, they appear to promote a localized destabilization to preserve activity.

The cold-adapted cellulase CelG has been purified from the culture supernatant of the Antarctic bacterium Pseudoalteromonas haloplanktis and the gene coding for this enzyme has been cloned, sequenced and expressed in Escherichia coli . This cellulase is composed of three structurally and functionally distinct regions: an N-terminal catalytic domain belonging to glycosidase family 5 and a C-terminal cellulose-binding domain belonging to carbohydrate-binding module family 5. The linker of 107 residues connecting both domains is one of the longest found in cellulases, and optimizes substrate accessibility to the catalytic domain by drastically increasing the surface of cellulose available to a bound enzyme molecule. The psychrophilic enzyme is closely related to the cellulase Cel5 from Erwinia chrysanthemi . Both k cat and k cat / K m values at 4 °C for the psychrophilic cellulase are similar to the values for Cel5 at 30–35 °C, suggesting temperature adaptation of the kinetic parameters. The thermodynamic parameters of activation of CelG suggest a heat-labile, relatively disordered active site with low substrate affinity, in agreement with the experimental data. The structure of CelG has been constructed by homology modelling with a molecule of cellotetraose docked into the active site. No structural alteration related to cold-activity can be found in the catalytic cleft, whereas several structural factors in the overall structure can explain the weak thermal stability, suggesting that the loss of stability provides the required active-site mobility at low temperatures.

Chloride-dependent alpha-amylases constitute a well conserved family of enzymes thereby allowing investigation of the characteristics of each member to understand, for example, relevant properties required for environmental adaptation. In this context, we have constructed a double mutant (Q58C/A99C) of the cold-active and heat-labile alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis, defined on the basis of its strong similarity with the mesophilic enzyme from pig pancreas. This mutant was characterized to understand the role of an extra disulfide bond specific to warm-blooded animals and located near the entrance of the catalytic cleft. We show that the catalytic parameters of the mutant are drastically modified and similar to those of the mesophilic enzyme. Calorimetric studies demonstrated that the mutant is globally stabilized (DeltaDeltaG = 1.87 kcal/mol at 20 degrees C) when compared with the wild-type enzyme, although the melting point (T(m)) was not increased. Moreover, fluorescence quenching experiments indicate a more compact structure for the mutated alpha-amylase. However, the strain imposed on the active site architecture induces a 2-fold higher thermal inactivation rate at 45 degrees C as well as the appearance of a less stable calorimetric domain. It is concluded that stabilization by the extra disulfide bond arises from an enthalpy-entropy compensation effect favoring the enthalpic contribution.

A colorimetric method for the determination of lipase activity in soil has been developed. Using p-nitrophenyl butyrate as substrate, soil samples are incubated at 30 °C and pH 7.25 for 10 min. After cooling on ice and centrifugation, the released p-nitrophenol is determined at 400 nm. To allow for the adsorption of p-nitrophenol onto soil, a calibration curve is prepared in the presence of soil.

The gene archb encoding for the cell-bound chitobiase from the Antarctic Gram-positive bacterium Arthrobacter sp. TAD20 was cloned and expressed in Escherichia coli in a soluble form. The mature chitobiase ArChb possesses four functionally independent domains. a catalytic domain stabilized by Ca2+, a,galactose-binding domain and an immunoglobulin-like domain followed by a cell-wall anchorage signal, typical of cell-surface proteins from Gram-positive bacteria. Binding of saccharides was analyzed by differential scanning calorimetry, allowing to distinguish unequivocally the catalytic domain from the galactose-binding domain and to study binding specificities. The results su,,est that ArChb could play a role in bacterium attachment to natural hosts. Kinetic parameters of ArChb demonstrate perfect adaptation to catalysis at low temperatures, as shown by a low activation energy associated with unusually low K-m and high k(cat) values. Thermodependence of these parameters indicates that discrete amino acid substitutions in the catalytic center have optimized the thermodynamic properties of weak interactions involved in substrate binding at low temperatures. Microcalorimetry also reveals that heat-lability, a general trait of psychrophilic enzymes, only affects the active site domain of ArChb. (C) 2001 Academic Press.

The heat-labile alpha-amylase from an antarctic bacterium is the largest known protein that unfolds reversibly according to a two-state transition as shown by differential scanning calorimetry. Mutants of this enzyme were produced, carrying additional weak interactions found in thermostable alpha-amylases. It is shown that single amino acid side chain substitutions can significantly modify the melting point T(m), the calorimetric enthalpy Delta H(cal), the cooperativity and reversibility of unfolding, the thermal inactivation rate constant, and the kinetic parameters k(cat) and K(m). The correlation between thermal inactivation and unfolding reversibility displayed by the mutants also shows that stabilizing interactions increase the frequency of side reactions during refolding, leading to intramolecular mismatches or aggregations typical of large proteins. Although all mutations were located far from the active site, their overall trend is to decrease both k(cat) and K(m) by rigidifying the molecule and to protect mutants against thermal inactivation. The effects of these mutations indicate that the cold-adapted alpha-amylase has lost a large number of weak interactions during evolution to reach the required conformational plasticity for catalysis at low temperatures, thereby producing an enzyme close to the lowest stability allowing maintenance of the native conformation.

The β-galactosidase from the Antarctic gram-negative bacterium Pseudoalteromonas haloplanktis TAE 79 was purified to homogeneity. The nucleotide sequence and the NH 2 -terminal amino acid sequence of the purified enzyme indicate that the β-galactosidase subunit is composed of 1,038 amino acids with a calculated M r of 118,068. This β-galactosidase shares structural properties with Escherichia coli β-galactosidase (comparable subunit mass, 51% amino sequence identity, conservation of amino acid residues involved in catalysis, similar optimal pH value, and requirement for divalent metal ions) but is characterized by a higher catalytic efficiency on synthetic and natural substrates and by a shift of apparent optimum activity toward low temperatures and lower thermal stability. The enzyme also differs by a higher pI (7.8) and by specific thermodynamic activation parameters. P. haloplanktis β-galactosidase was expressed in E. coli , and the recombinant enzyme displays properties identical to those of the wild-type enzyme. Heat-induced unfolding monitored by intrinsic fluorescence spectroscopy showed lower melting point values for both P. haloplanktis wild-type and recombinant β-galactosidase compared to the mesophilic enzyme. Assays of lactose hydrolysis in milk demonstrate that P. haloplanktis β-galactosidase can outperform the current commercial β-galactosidase from Kluyveromyces marxianus var. lactis, suggesting that the cold-adapted β-galactosidase could be used to hydrolyze lactose in dairy products processed in refrigerated plants.

A total of 59 bacteria samples from Antarctic sea water were collected and screened for their ability to produce α-amylase. The highest activity was recorded from an isolate identified as an Alteromonas species. The purified α-amylase shows a molecular mass of about 50 000 Da and a pI of 5.2. The enzyme is stable from pH 7.5 to 9 and has a maximal activity at pH 7.5. Compared with other α-amylases from mesophiles and thermophiles, the "cold enzyme" displays a higher activity at low temperature and a lower stability at high temperature. The psychrophilic α-amylase requires both Cl-and Ca2+for its amylolytic activity. Br-is also quite effecient as an allosteric effector. The comparison of the amino acid composition with those of other α-amylases from various organisms shows that the cold α-amylase has the lowest content in Arg and Pro residues. This could be involved in the principle used by the psychrophilic enzyme to adapt its molecular structure to the low temperature of the environment. Key words: α-amylase, psychrophilic microorganisms, Antarctic.

The thermal stability of the cold-active α-amylase (AHA) secreted by the Antarctic bacterium Alteromonas haloplanctis has been investigated by intrinsic fluorescence, circular dichroism, and differential scanning calorimetry. It was found that this heat-labile enzyme is the largest known multidomain protein exhibiting a reversible two-state unfolding, as demonstrated by the recovery of ΔHcal values after consecutive calorimetric transitions, a ΔHcal/ΔHeff ratio close to unity, and the independence of unfolding thermodynamic parameters of scan rates. By contrast, the mesophilic α-amylases investigated here (from porcine pancreas, human salivary glands, yellow meal beetle, Bacillus amyloliquefaciens, and Bacillus licheniformis) unfold irreversibly according to a non-two-state mechanism. Unlike mesophilic α-amylases, the melting point of AHA is independent of calcium and chloride binding while the allosteric and structural functions of these ions are conserved. The thermostability of AHA at optimal condition...

We performed a comparative study on the enzymological features of purified recombinant α-amylase of three species belonging to the Drosophila melanogaster species subgroup: D. melanogaster, D. erecta and D. sechellia. D. erecta an d D. sechellia are specialist species, with host plant Pandanus candelabrum (Pandanaceae) and M orinda citrifolia (Rubiaceae), respectively. The temperature o ptima were around 57–60°C for the three species. The pH optima were 7.2 for D. melanogaster, 8.2 for D. erecta and 8.5 for D. sechellia. The kcat and Km were also estimated for each species with different substrates. The specialist species D. erecta and D. sechellia display a higher affinity for starch than D. melanogaster. α-Amylase activity is higher on starch than on glycogen in all species. α-A mylases of D. erecta and D. sechellia have a higher activity on maltooligosaccharides (G6 and G7) than on starch, contrary to D. melanogaster. Such differences in the e nzymological features between the species might reflect adaptation to different ecological niches and feeding habits.

The lipase produced by Burkholderia glumae folds spontaneously into an inactive near-native state and requires a periplasmic chaperone to reach its final active and secretion-competent fold. The B. glumae lipase-specific foldase (Lif) is classified as a member of the steric-chaperone family of which the propeptides of alpha-lytic protease and subtilisin are the best known representatives. Steric chaperones play a key role in conferring kinetic stability to proteins. However, until present there was no solid experimental evidence that Lif-dependent lipases are kinetically trapped enzymes. By combining thermal denaturation studies with proteolytic resistance experiments and the description of distinct folding intermediates, we demonstrate that the native lipase has a kinetically stable conformation. We show that a newly discovered molten globule-like conformation has distinct properties that clearly differ from those of the near-native intermediate state. The folding fingerprint of Lif-dependent lipases is put in the context of the protease-prodomain system and the comparison reveals clear differences that render the lipase-Lif systems unique. Limited proteolysis unveils structural differences between the near-native intermediate and the native conformation and sets the stage to shed light onto the nature of the kinetic barrier.

The lack of hemoglobin and of carbonic anhydrase in the blood of icefish suggest that substantial adaptations of the acid-base balance should occur in order to ensure blood pH homeostasis. The level of peptidic histidyl and of reactive -SH groups per unit of body mass in icefish plasma are 12–13 times higher than those of Notothenia rossii , a common red-blooded Antarctic species. It is proposed that the high level of imidazole ring in icefish plasma improves the non-bicarbonate buffering capacity and that the reactive sulfhydryls are involved in a redox buffer as in some other hypoxia tolerant species. After plasma fractionation on Ultrogel AcA 34, the two main icefish serum proteins have been purified by DEAE cellulose chromatography (IFI) and by HPLC on anion exchange column (IF2). IFI has been identified as a cysteine-rich para-albumin and IF2 as an histidine-rich immunoglobulin-like protein.

The mutants Mut5 and Mut5CC from a psychrophilic α-amylase bear representative stabilizing interactions found in the heat-stable porcine pancreatic α-amylase but lacking in the cold-active enzyme from an Antarctic bacterium. From an evolutionary perspective, these mutants can be regarded as structural intermediates between the psychrophilic and the mesophilic enzymes. We found that these engineered interactions improve all the investigated parameters related to protein stability as follows: compactness; kinetically driven stability; thermodynamic stability; resistance toward chemical denaturation, and the kinetics of unfolding/refolding. Concomitantly to this improved stability, both mutants have lost the kinetic optimization to low temperature activity displayed by the parent psychrophilic enzyme. These results provide strong experimental support to the hypothesis assuming that the disappearance of stabilizing interactions in psychrophilic enzymes increases the amplitude of concerted motions required by catalysis and the dynamics of active site residues at low temperature, leading to a higher activity.

The proteomes expressed at 4 degrees C and 18 degrees C by the psychrophilic Antarctic bacterium Pseudoalteromonas haloplanktis have been compared using two-dimensional differential in-gel electrophoresis, showing that translation, protein folding, membrane integrity and anti-oxidant activities are upregulated at 4 degrees C. This proteomic analysis revealed that the trigger factor is the main upregulated protein at low temperature. The trigger factor is the first molecular chaperone interacting with virtually all newly synthesized polypeptides on the ribosome and also possesses a peptidyl-prolyl cis-trans isomerase activity. This suggests that protein folding at low temperatures is a rate-limiting step for bacterial growth in cold environments. It is proposed that the psychrophilic trigger factor rescues the chaperone function as both DnaK and GroEL (the major bacterial chaperones but also heat-shock proteins) are downregulated at 4 degrees C. The recombinant psychrophilic trigger factor is a monomer that displays unusually low conformational stability with a Tm value of 33 degrees C, suggesting that the essential chaperone function requires considerable flexibility and dynamics to compensate for the reduction of molecular motions at freezing temperatures. Its chaperone activity is strongly temperature-dependent and requires near-zero temperature to stably bind a model-unfolded polypeptide.

The crystal structure of a cold-active aminopeptidase (ColAP) from Colwellia psychrerythraea strain 34H has been determined, extending the number of crystal structures of the M1 metallopeptidase family to four among the 436 members currently identified. In agreement with their sequence similarity, the overall structure of ColAP displayed a high correspondence with leukotriene A4 hydrolase (LTA4H), a human bifunctional enzyme that converts leukotriene A4 (LTA4) in the potent chemoattractant leukotriene B4. Indeed, both enzymes are composed of three domains, an N-terminal saddle-like domain, a catalytic thermolysin-like domain, and a less conserved C-terminal alpha-helical flat spiral domain. Together, these domains form a deep cavity harboring the zinc binding site formed by residues included in the conserved HEXXHX(18)H motif. A detailed structural comparison of these enzymes revealed several plausible determinants of ColAP cold adaptation. The main differences involve specific amino acid substitutions, loop content and solvent exposure, complexity and distribution of ion pairs, and differential domain flexibilities. Such elements may act synergistically to allow conformational flexibility needed for an efficient catalysis in cold environments. Furthermore, the region of ColAP corresponding to the aminopeptidase active site of LTA4H is much more conserved than the suggested LTA4 substrate binding region. This observation supports the hypothesis that this region of the LTA4H active site has evolved in order to fit the lipidic substrate.

Tropomyosin of fast-twitch, slow-twitch and cardiac muscles of carp and icefish has been isolated by hydroxyapatite chromatography. The subunit distribution has been investigated by polyacrylamide gel electrophoresis and by peptide mapping. The purified skeletal muscle tropomyosins all belong to the alpha family and differ from higher vertebrate tropomyosin by the lack of beta subunits. Specific alpha isotypes are however encountered in fast-twitch fibres (alpha w subunit) and slow-twitch or intermediate (pink) fibres (alpha and alpha w subunits). The amino acid compositions and the paracrystals formed by the carp alpha w alpha w and alpha alpha w tropomyosins do not differ markedly from that of rabbit alpha alpha chains. They differ however by their capability to inhibit the ATPase activity of rabbit skeletal muscle acto-HMM system. A beta-like subunit is found in carp cardiac tropomyosin, in the proportion of 25% of the native protein, but not in icefish heart.

The structures of inactive mutants D144A and E78Q of the glycoside hydrolase family 8 (GH-8) endo-beta-1,4-d-xylanase (pXyl) from the Antarctic bacterium Pseudoalteromonas haloplanktis TAH3a in complex with its substrate xylopentaose (at 1.95 A resolution) and product xylotriose (at 1.9 A resolution) have been determined by X-ray crystallography. A detailed comparative analysis of these with the apo-enzyme and with other GH-8 structures indicates an induced fit mechanism upon ligand binding whereby a number of conformational changes and, in particular, a repositioning of the proton donor into a more catalytically competent position occurs. This has also allowed for the description of protein-ligand interactions in this enzyme and for the demarcation of subsites -3 to +3. An in-depth analysis of each of these subsites gives an insight into the structure-function relationship of this enzyme and the basis of xylose/glucose discrimination in family 8 glycoside hydrolases. Furthermore, the structure of the -1/+1 subsite spanning complex reveals that the substrate is distorted from its ground state conformation. Indeed, structural analysis and in silico docking studies indicate that substrate hydrolysis in GH-8 members is preceded by a conformational change, away from the substrate ground-state chair conformation, to a pretransition state local minimum (2)S(O) conformation.

A new microcalorimetric method for recording the kinetic parameters k(cat), K(m) and K(i) of alpha-amylases using polysaccharides and oligosaccharides as substrates is described. This method is based on the heat released by glycosidic bond hydrolysis. The method has been developed to study the active site properties of the cold-active alpha-amylase produced by an Antarctic psychrophilic bacterium in comparison with its closest structural homolog from pig pancreas. It is shown that the psychrophilic alpha-amylase is more active on large macromolecular substrates and that the higher rate constants k(cat) are gained at the expense of a lower affinity for the substrate. The active site is able to accommodate larger inhibitory complexes, resulting in a mixed-type inhibition of starch hydrolysis by maltose. A method for recording the binding enthalpies by isothermal titration calorimetry in a low-affinity system has been developed, allowing analysis of the energetics of weak ligand binding using the allosteric activator chloride. It is shown that the low affinity of the psychrophilic alpha-amylase for chloride is entropically driven. The high enthalpic and entropic contributions of activator binding suggest large structural fluctuations between the free and the bound states of the cold-active enzyme. The kinetic and thermodynamic data for the psychrophilic alpha-amylase indicate that the strictly conserved side-chains involved in substrate binding and catalysis possess an improved mobility, responsible for activity in the cold, and resulting from the disappearance of stabilizing interactions far from the active site.

Site-directed mutagenesis and a comparative characterisation of the kinetic parameters, pH dependency of activity and thermal stability of mutant and wild-type enzymes have been used in association with crystallographic analysis to delineate the functions of several active site residues in a novel glycoside hydrolase family 8 xylanase. Each of the residues investigated plays an essential role in this enzyme: E78 as the general acid, D281 as the general base and in orientating the nucleophilic water molecule, Y203 in maintaining the position of the nucleophilic water molecule and in structural integrity and D144 in sugar ring distortion and transition state stabilization. Interestingly, although crystal structure analyses and the pH-activity profiles clearly identify the functions of E78 and D281, substitution of these residues with their amide derivatives results in only a 250-fold and 700-fold reduction in their apparent k cat values, respectively. This, in addition to the observation that the proposed general base is not conserved in all glycoside hydrolase family 8 enzymes, indicates that the mechanistic architecture in this family of inverting enzymes is more complex than is conventionally believed and points to a diversity in the identity of the mechanistically important residues as well as in the arrangement of the intricate microenvironment of the active site among members of this family.

International audience; Amylosucrase from Neisseria polysaccharea is a remarkable transglucosidase from family 13 of the glycoside-hydrolases that synthesizes an insoluble amylose-like polymer from sucrose in the absence of any primer. Amylosucrase shares strong structural similarities with alpha-amylases. Exactly how this enzyme catalyzes the formation of alpha-1,4-glucan and which structural features are involved in this unique functionality existing in family 13 are important questions still not fully answered. Here, we provide evidence that amylosucrase initializes polymer formation by releasing, through sucrose hydrolysis, a glucose molecule that is subsequently used as the first acceptor molecule. Maltooligosaccharides of increasing size were produced and successively elongated at their nonreducing ends until they reached a critical size and concentration, causing precipitation. The ability of amylosucrase to bind and to elongate maltooligosaccharides is notably due to the presence of key residues at the OB1 acceptor binding site that contribute strongly to the guidance (Arg415, subsite +4) and the correct positioning (Asp394 and Arg446, subsite +1) of acceptor molecules. On the other hand, Arg226 (subsites +2/+3) limits the binding of maltooligosaccharides, resulting in the accumulation of small products (G to G3) in the medium. A remarkable mutant (R226A), activated by the products it forms, was generated. It yields twice as much insoluble glucan as the wild-type enzyme and leads to the production of lower quantities of by-products.

DNA ligases are important enzymes required for cellular processes such as DNA replication, recombination, and repair. NAD(+)-dependent DNA ligases are essentially restricted to eubacteria, thus constituting an attractive target in the development of novel antibiotics. Although such a project might involve the systematic testing of a vast number of chemical compounds, it can essentially gain from the preliminary deciphering of the conformational stability and structural perturbations associated with the formation of the catalytically active adenylated enzyme. We have, therefore, investigated the adenylation-induced conformational changes in the mesophilic Escherichia coli and thermophilic Thermus scotoductus NAD(+)-DNA ligases, and the resistance of these enzymes to thermal and chemical (guanidine hydrochloride) denaturation. Our results clearly demonstrate that anchoring of the cofactor induces a conformational rearrangement within the active site of both mesophilic and thermophilic enzymes accompanied by their partial compaction. Furthermore, the adenylation of enzymes increases their resistance to thermal and chemical denaturation, establishing a thermodynamic link between cofactor binding and conformational stability enhancement. Finally, guanidine hydrochloride-induced unfolding of NAD(+)-dependent DNA ligases is shown to be a complex process that involves accumulation of at least two equilibrium intermediates, the molten globule and its precursor.

Adapting metabolic enzymes of microorganisms to low temperature environments may require a difficult compromise between velocity and affinity. We have investigated catalytic efficiency in a key metabolic enzyme (dihydrofolate reductase) of Moritella profunda sp. nov., a strictly psychrophilic bacterium with a maximal growth rate at 2°C or less. The enzyme is monomeric ( M r = 18,291), 55% identical to its Escherichia coli counterpart, and displays T m and denaturation enthalpy changes much lower than E. coli and Thermotoga maritima homologues. Its stability curve indicates a maximum stability above the temperature range of the organism, and predicts cold denaturation below 0°C. At mesophilic temperatures the apparent K m value for dihydrofolate is 50- to 80-fold higher than for E. coli , Lactobacillus casei , and T. maritima dihydrofolate reductases, whereas the apparent K m value for NADPH, though higher, remains in the same order of magnitude. At 5°C these values are not significantly modified. The enzyme is also much less sensitive than its E. coli counterpart to the inhibitors methotrexate and trimethoprim. The catalytic efficiency ( k cat /K m ) with respect to dihydrofolate is thus much lower than in the other three bacteria. The higher affinity for NADPH could have been maintained by selection since NADPH assists the release of the product tetrahydrofolate. Dihydrofolate reductase adaptation to low temperature thus appears to have entailed a pronounced trade-off between affinity and catalytic velocity. The kinetic features of this psychrophilic protein suggest that enzyme adaptation to low temperature may be constrained by natural limits to optimization of catalytic efficiency.

The gene encoding chitinase ArChiB from the Antarctic Arthrobacter sp. TAD20 has been expressed in Escherichia coli and the recombinant enzyme purified to homogeneity. In an effort to engineer cold-adapted biocatalysts through rational redesign to operate at elevated temperatures, we performed several mutations aiming to increase the rigidity of the molecular edifice of the selected psychrophilic chitinase. The mutations were designed on the basis of a homology-based three-dimensional model of the enzyme, and included an attempt to introduce a salt bridge (mutant N198K) and replacements of selected Gly residues by either Pro (mutants G93P, G254P) or Gln (G406Q). Mutant N198K resulted in a more stable protein (DeltaTm = 0.6 degrees C). Mutant G93P exhibited a DeltaTm of 1.2 degrees C, while mutants G254P and G406Q exhibited decreased stability. We conclude that the effect of mutating Gly residues on enzyme stability is rather complex and can only be understood in the context of the structural environment. Kinetic and spectroscopic analysis of these enzyme variants revealed that the kinetic parameters kcat and Km have been significantly modified.

The alpha-amylase precursor from the bacterium Pseudoalteromonas haloplanktis possesses a propeptide at the C-terminus possibly responsible for outer membrane translocation. Unlike the predicted beta-barrel of autotransporters, this C-terminal propeptide displays a noticeable alpha-helix content. It is connected to the enzyme by a disordered linker and has no significant interaction with the catalytic domain. The microcalorimetric pattern of the precursor also demonstrates that the stability of protein domains may evolve differently.

Psychrophiles, host of permanently cold habitats, display metabolic fluxes comparable to those exhibited by mesophilic organisms at moderate temperatures. These organisms have evolved by producing, among other peculiarities, cold-active enzymes that have the properties to cope with the reduction of chemical reaction rates induced by low temperatures. The emerging picture suggests that these enzymes display a high catalytic efficiency at low temperatures through an improved flexibility of the structural components involved in the catalytic cycle, whereas other protein regions, if not implicated in catalysis, may be even more rigid than their mesophilic counterparts. In return, the increased flexibility leads to a decreased stability of psychrophilic enzymes. In order to gain further advances in the analysis of the activity/flexibility/stability concept, psychrophilic, mesophilic, and thermophilic DNA ligases have been compared by three-dimensional-modeling studies, as well as regards their activity, surface hydrophobicity, structural permeability, conformational stabilities, and irreversible thermal unfolding. These data show that the cold-adapted DNA ligase is characterized by an increased activity at low and moderate temperatures, an overall destabilization of the molecular edifice, especially at the active site, and a high conformational flexibility. The opposite trend is observed in the mesophilic and thermophilic counterparts, the latter being characterized by a reduced low temperature activity, high stability and reduced flexibility. These results strongly suggest a complex relationship between activity, flexibility and stability. In addition, they also indicate that in cold-adapted enzymes, the driving force for denaturation is a large entropy change.

The Antarctic psychrophile Pseudoalteromonas haloplanktis produces a cold-active cellulase. To date, a three-dimensional structure of a psychrophilic cellulase has been lacking. Crystallographic studies of this cold-adapted enzyme have therefore been initiated in order to contribute to the understanding of the molecular basis of the cold adaptation and the high catalytic efficiency of the enzyme at low and moderate temperatures. The catalytic core domain of the psychrophilic cellulase CelG from P. haloplanktis has been expressed, purified and crystallized and a complete diffraction data set to 1.8 A has been collected. The space group was found to be P2(1)2(1)2(1), with unit-cell parameters a = 135.1, b = 78.4, c = 44.1 A. A molecular-replacement solution, using the structure of the mesophilic counterpart Cel5A from Erwinia chrysanthemi as a search model, has been found.

Five psychrophilic Antarctic bacteria have been selected for their capacity to secrete exoenzymes into culture medium. These strains are able to grow from 0 to about 25° C. However, production of lipase fromMoraxella, α-amylase fromAlteromonas haloplanctis, β-lactamase fromPsychrobacter immobilis and protease fromBacillus is maximal at temperatures close to that of their environment (—2 to 4° C) and is strongly inhibited at higher temperatures. This thermal effect involves alterations in the secretory pathway in the upper range of temperatures, losses due to the enzyme thermal lability and in some cases to reduction in cell development. The apparent optimal activity temperature of these enzymes is between 30 and 40° C, i.e. about 20° C lower than that of their mesophilic counterparts.

Secretion of cold-adapted α-amylase from Pseudoalteromonas haloplanktis TAB23 was studied in three Antarctic bacteria. We demonstrated that the enzyme is specifically secreted in the psychrophilic hosts even in the absence of a protein domain that has been previously reported to be necessary for α-amylase secretion in Escherichia coli . The occurrence of two different secretion pathways in different hosts is proposed.

Xylanases are generally classified into glycosyl hydrolase families 10 and 11 and are found to frequently have an inverse relationship between their pI and molecular mass values. However, we have isolated a psychrophilic xylanase that belongs to family 8 and which has both a high pI and high molecular mass. This novel xylanase, isolated from the Antarctic bacterium Pseudoalteromonas haloplanktis, is not homologous to family 10 or 11 enzymes but has 20-30% identity with family 8 members. NMR analysis shows that this enzyme hydrolyzes with inversion of anomeric configuration, in contrast to other known xylanases which are retaining. No cellulase, chitosanase or lichenase activity was detected. It appears to be functionally similar to family 11 xylanases. It hydrolyzes xylan to principally xylotriose and xylotetraose and is most active on long chain xylo-oligosaccharides. Kinetic studies indicate that it has a large substrate binding cleft, containing at least six xylose-binding subsites. Typical psychrophilic characteristics of a high catalytic activity at low temperatures and low thermal stability are observed. An evolutionary tree of family 8 enzymes revealed the presence of six distinct clusters. Indeed classification in family 8 would suggest an (alpha/alpha)(6) fold, distinct from that of other currently known xylanases.

Isothermal titration calorimetry has been applied to the determination of the kinetic parameters of chitinases (EC 3.2.1.14) by monitoring the heat released during the hydrolysis of chitin glycosidic bonds. Experiments were carried out using two different macromolecular substrates: a soluble polymer of N-acetylglucosamine and the insoluble chitin from crab shells. Different experimental temperatures were used in order to compare the thermodependence of the activity of two chitinases from the psychrophile Arthrobacter sp. TAD20 and of chitinase A from the mesophile Serratia marcescens. The method allowed to determine unequivocally the catalytic rate constant k(cat), the activation energy (E-a) and the thermodynamic activation parameters (DeltaG(#), DeltaH(#), DeltaS(#)) of the chitinolytic reaction on the soluble substrate. The catalytic activity has also been determined on insoluble chitin, which displays an effect of substrate saturation by chitinases. On both substrates, the thermodependence of the activity of the psychrophilic chitinases was lower than that observed with the mesophilic counterpart. (C) 2001 Elsevier Science B.V. All rights reserved.

The gene encoding the phosphoglycerate kinase (PGK) from the Antarctic Pseudomonas sp. TACII18 has been cloned and found to be inserted between the genes encoding for glyceraldhyde-3-phosphate dehydrogenase and fructose aldolase. The His-tagged and the native recombinant PGK from the psychrophilic Pseudomonas were expressed in Escherichia coli. The wild-type and the native recombinant enzymes displayed identical properties, such as a decreased thermostability and a 2-fold higher catalytic efficiency at 25 degrees C when compared with the mesophilic PGK from yeast. These properties, which reflect typical features of cold-adapted enzymes, were strongly altered in the His-tagged recombinant PGK. The structural model of the psychrophilic PGK indicated that a key determinant of its low stability is the reduced number of salt bridges, surface charges, and aromatic interactions when compared with mesophilic and thermophilic PGK. Differential scanning calorimetry of the psychrophilic PGK revealed unusual variations in its conformational stability for the free and substrate-bound forms. In the free form, a heat-labile and a thermostable domain unfold independently. It is proposed that the heat-labile domain acts as a destabilizing domain, providing the required flexibility around the active site for catalysis at low temperatures.

The alpha-amylase sequences contained in databanks were screened for the presence of amino acid residues Arg195, Asn298 and Arg/Lys337 forming the chloride-binding site of several specialized alpha-amylases allosterically activated by this anion. This search provides 38 alpha-amylases potentially binding a chloride ion. All belong to animals, including mammals, birds, insects, acari, nematodes, molluscs, crustaceans and are also found in three extremophilic Gram-negative bacteria. An evolutionary distance tree based on complete amino acid sequences was constructed, revealing four distinct clusters of species. On the basis of multiple sequence alignment and homology modeling, invariable structural elements were defined, corresponding to the active site, the substrate binding site, the accessory binding sites, the Ca(2+) and Cl(-) binding sites, a protease-like catalytic triad and disulfide bonds. The sequence variations within functional elements allowed engineering strategies to be proposed, aimed at identifying and modifying the specificity, activity and stability of chloride-dependent alpha-amylases.

Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the alpha-amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known alpha-amylases of various origins with a surprisingly greatest similarity to mammalian alpha-amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds. The chloride binding site is situated approximately 5 angstroms from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found approximately 22 angstroms from the active site. We found that this triad is equally present in other chloride dependent alpha-amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted alpha-amylase.

α-Amylase from the antarctic psychrophile Alteromonas haloplanktis is synthesized at 0 ± 2°C by the wild strain. This heat-labile α-amylase folds correctly when overexpressed in Escherichia coli , providing the culture temperature is sufficiently low to avoid irreversible denaturation. In the described expression system, a compromise between enzyme stability and E. coli growth rate is reached at 18°C.